Most human inherited diseases and cancers are known to be caused by mutations in nuclear genes. In general, a mutation is considered to be particular polymorphic variants at a genetic locus. The mutation can be a single nucleotide difference, often referred to as a point mutation. Determination of mutational spectra in disease-related genes in non-tumor tissues can provide direct evidence as to whether a specific mutagenic agent or pathway is involved in a particular human disease. In the field of genetic epidemiology, for example, it is useful to be able to detect patterns in the frequencies of genetic polymorphisms (e.g., mutations) that are indicative of the causes of disease. In the field of pediatric genetics, detection of polymorphisms is useful to screen for early diagnosis of rare genetic diseases in newborns. In genetic counseling of prospective parents, detection of polymorphisms in their cells is anticipated to be of significant predictive value in determining the quality of life for newborns. In addition, the detection of polymorphisms can be useful in the development of pharmaceuticals, such as vaccines or recombinant proteins. The detection of polymorphisms is also useful in toxicological studies to determine if genetic damage has occurred due to specific agents, such as additives in cosmetics or environmental contaminants.
At the cellular and tissue level, polymorphisms at a specific genetic locus may give rise to significantly altered cellular behavior. However, because even relatively small cell or tissue samples can contain millions or billions of DNA molecules containing the particular genetic locus, an examination of the mutational spectra, a representation of the range and frequencies of polymorphic variants at a genetic locus, requires detecting alleles that are potentially present at a very low frequency. In fact, since many pathological conditions manifest themselves even where only a small fraction of the DNA is mutated, it is probable that detection of the rare alleles is important for the early detection of many pathological disorders. For example, the ability to detect cancer cells by virtue of a polymorphism present in a small fraction of cells within a tissue or blood sample can be useful to detect metastasis of the cancer, to use as a signal that the cancer is recurring, or as a screen for the initial appearance of a cancer. Additionally, determining the mutational spectra of, for example, the tumor suppressor gene, p53, in non-tumor tissue of a tumor bearing organ may lead to identification of the probable cause of a tumor (Harris, C., 1993. Science, 262:1980–1981).
A number of methods have been used to detect mutant DNA sequences, including isolation of DNA from cells, cloning and sequencing the cloned product. Several electrophoretic methods have been used to separate mutant DNA from wild-type DNA including, for example, denaturing gradient gel electrophoresis (DGGE) (Fischer, S. and Lerman, L., 1983. Proc. Natl. Acad. Sci. USA., 80:1579–1583; Cariello, N. et al., 1988. Am. J. Hum. Genet., 42:726–734). However, these methods are tedious and difficult to use. Further, certain common laboratory practices, such as labeling DNA molecules with radioactive phosphorous, create radiolysis reactions that interfere with these methods. Interfering reaction products also arise due to thermolysis in separation extending for many hours at a temperature over 60° C., photochemical reactions with light from ordinary laboratory fluorescent fixtures, and from chemical reactions that presumably involve active oxygen species present in aqueous solutions.
As methods for detecting polymorphisms currently known in the art do not allow for the detection of rare polymorphisms in relatively small samples, the present state of the art can not allow for detection of rare mutations in, for example, stem cells. Thus, a fast and reproducible method that can detect mutant DNA sequences present in a sample, including mutant DNA sequences that occur as a small fraction of DNA molecules relative to the total number of DNA molecules present in a sample, would be very important.